Mycobacterium bacteriophage bactericidal protein and preparation method thereof

Abstract

The invention discloses mycobacterium bacteriophage bactericidal protein and a preparation method thereof. The preparation method comprises the following steps: S1, constructing a bacteriophage gene gp48 induced expression strain; s2, determining a phage gene expression strain; s3, constructing a phage metallophosphatase induced expression strain; s4, purifying the phage metal phosphatase protein; according to the invention, a metal phosphoesterase is identified in a bacteriophage genome with relatively strong lysis activity on mycobacterium tuberculosis and mycobacterium smegmatis, and the expression of the metal phosphoesterase has relatively strong killing activity on bacteria and relatively strong phosphodiesterase activity in vitro; the invention also provides a safe and convenient protein preparation method of the phage metal phosphatase.

Description

Technical Field

[0001] The present invention belongs to the technical field of mycobacterial bactericidal proteins, and particularly relates to a mycobacteriophage bactericidal protein and a preparation method thereof. Background Art

[0002] Mycobacterium is a genus of Actinomycetes, which is divided into pathogenic mycobacteria and non-pathogenic mycobacteria. Mycobacterium tuberculosis is a pathogenic mycobacterium and is the pathogen of tuberculosis. Tuberculosis is usually treated by combining multiple antibiotics to kill Mycobacterium tuberculosis. However, Mycobacterium tuberculosis is extremely prone to drug resistance due to its special extracellular components. Therefore, phages have been widely concerned as a new strategy to kill Mycobacterium tuberculosis. During the process of infecting bacteria, phages inject their own genomes into host cells and express the proteins they need through a series of transcription and translation processes. Finally, they complete replication, assemble into mature progeny phage particles, lyse the host cells, and release the progeny. The most important step in this whole process is how phages inhibit and kill the host through their own proteins and hijack the resources of the host cells to complete their own life activities. For phages of other pathogenic or non-pathogenic bacteria such as Escherichia coli phage, Pseudomonas aeruginosa phage, and Staphylococcus aureus phage, the antibacterial or bactericidal proteins encoded by themselves are relatively clear. However, the overall research on mycobacteriophages is very limited, and the identified antibacterial genes or bactericidal genes are also very few. For the identified mycobacterial antibacterial or bactericidal genes, their effects have not been systematically detected, and there is no relatively mature method for their expression and purification. The identification of mycobacteriophage antibacterial or bactericidal proteins can help us better understand the bactericidal mechanism of phages and can also be applied to the treatment of diseases caused by pathogenic mycobacteria.

[0003] Regarding the in vitro purification of phage-related proteins, most of them also focus on other phages, and there are almost no reports on the purification methods of mycobacteriophage proteins. In recent years, the existing experimental methods for purifying the metal phosphatase encoded by mycobacteriophages in the prior art cannot be repeated subsequently. There is a relative lack of research on the screening and identification of mycobacteriophage antibacterial or bactericidal proteins and the purification methods of mycobacteriophage proteins. Summary of the Invention

[0004] The purpose of the present invention is to provide a mycobacteriophage bactericidal protein and a preparation method thereof. A metal phosphatase was identified in the genome of a phage with strong lytic activity against Mycobacterium tuberculosis and Mycobacterium smegmatis. Its expression has strong bactericidal activity against bacteria and strong phosphodiesterase activity in vitro. And a safe and convenient protein preparation method for phage metal phosphatase is provided.

[0005] The present invention is achieved through the following technical solutions: A mycobacteriophage bactericidal protein, comprising a gene gp48 , and the gp48 base sequence of the gene is shown as SEQ ID No.1.

[0006] Furthermore: The amino acid sequence is shown as SEQ ID No.2.

[0007] A preparation method of a mycobacteriophage bactericidal protein, comprising the following steps: S1: Construct a phage gene gp48 induced expression strain; S2: Measure the growth of the phage gene expression strain; S3: Construct a phage metallophosphatase induced expression strain; S4: Purify the phage metallophosphatase protein.

[0008] Furthermore, the specific steps of constructing the phage gene gp48 induced expression strain include: S11: Design a primer pair for the gene gp48 with the A10ZJ24 phage genome as a template and perform amplification; S12: Obtain the recombinant plasmid pJR962 and transform it into mycobacteria; S13: Perform PCR verification.

[0009] Furthermore, the primer pair for the gene gp48 is: gp48-F: ATAGAGAAGGCGGTATCGGTGACCGAGCGCATCGTCGT; gp48-R: CTTAGCTAATCAGCGGCCGCTCAGACCTCCCAAGTGTGACCGTCT.

[0010] Furthermore, the specific method for obtaining the recombinant plasmid pJR962 is: Using seamless cloning technology, react at 50 °C for 30 minutes, and ligate the target fragment to the ClaI-NotI site on the linearized pJR962 induction vector to obtain the recombinant plasmid pJR962.

[0011] Furthermore, the specific steps of constructing the phage metallophosphatase induced expression strain include: S31: Using the A10ZJ24 phage genome as a template, design a primer pair for phage metallophosphatase expression and amplify the gene gp48; S32: Obtain the recombinant plasmid pET28a-SUMO and transform it into Escherichia coli; S33: Conduct PCR verification and sequencing verification.

[0012] Furthermore, the primer pair for expressing the phage metallophosphatase is: Gp48Exp-F: GGATCCGTGACCGAGCGCATCGTCGT; Gp48Exp-R: AAGCTTTCAGACCTCCCAAGTGTGAC.

[0013] Furthermore, the purification of the phage metallophosphatase protein includes the following steps: S41: Inoculate the verified strain into a medium for culture, centrifuge, and collect the bacterial cells; S42: Disrupt, bind to the column, elute the collected bacterial cells, and obtain the target protein; S43: Detect the target protein, perform dialysis, and then store it.

[0014] Furthermore, when detecting the target protein, the band size and purity are detected by polyacrylamide gel electrophoresis.

[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1) In the present invention, a metallophosphatase was identified in the genome of a phage with strong lytic activity against Mycobacterium tuberculosis and Mycobacterium smegmatis. Its expression has strong killing activity against bacteria and strong phosphodiesterase activity in vitro; and a safe and convenient method for preparing the phage metallophosphatase protein is provided.

[0016] 2) In the present invention, the gp48 metallophosphatase encoded by the gene is a novel mycobacteriophage bactericidal protein, and its expression will have a killing effect on both pathogenic and non-pathogenic mycobacteria.

[0017] 3) In the present invention, a protein with relatively high purity can be obtained through the purification method of the phage metallophosphatase, and this protein has strong phosphodiesterase activity in vitro. Description of the Drawings

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0019] Figure 1 Schematic diagram of the location of the metallophosphatase encoded by the phage in the present invention.

[0020] Figure 2 PCR verification result of the pJR962 inducible expression vector in the present invention.

[0021] Figure 3 In the present invention gp48 Comparison diagram of the inducible expression strain before and after induction on a 7H10 solid plate.

[0022] Figure 4 In the present invention gp48 Comparison diagram of the inducible expression strain before and after induction in 7H9 liquid medium.

[0023] Figure 5 PCR verification result diagram of the pET28a-SUMO inducible expression vector in the present invention.

[0024] Figure 6 SDS-PAGE result diagram of the purified phage metallophosphatase in the present invention.

[0025] Figure 7 Phosphodiesterase activity assay result diagram of the phage metallophosphatase in the present invention. Detailed implementation manners

[0026] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention.

[0027] Example 1: A mycobacteriophage bactericidal protein, including gene gp48 gene gp48 The base sequence of which is shown in SEQ ID No.1: >A10ZJ24 gp48 1 GTGACCGAGC GCATCGTCGT CATCAGCGAC ACCCAGATCC CTTACGACGA CCGGCGTGCC 61 CTGCGGGCCG TCATCCGGTT CATCGGGGAC TACCAGCCCA CCAGGGTCAT CCACATCGGT 121 GACCTGATGG ACTTCCCGCA ACCGTCCCGC TGGAACAAGG ACACTCGCGG TGAGTTCGAG 181 GGCTCGGTGT TCAAGGATGC AGAGCAGTGC AAGCGTCGTT TCCTCGATCC GCTGCGGACC 241 GTGTACGTCG GTCCGGTCGG TGTCCATGAG GGCAACCACG ACGAGCGTCC TCGGACCTAC 301 CTGGCCAAGT ACGCGCCGGC CTTGGCCGAG TCTCGGGCAT TTCACTTCGA GAACCTGCTC 361 GACTTCGACG GGTACGGCAT CGAGGTGCTG CCTGAGTTCT ACAAGGTCGC ACCGGGCTGG 421 GTGACCACCC ATGGGCACAG GGGCCAGATC AGCCTGTCGC GGATCGCCGG TAACACGGCG 481 CTCAACGCGG CACGGAAGTT CGGGACCTCG GTCGTCATGG GCCACACCCA CCGGCTGGGC 541 ATCGGGAGCC ACACCGAGGG CTACGGCGGC ATCTCCAAGC GGGTCCTGAC CGGCATGGAG 601 GTCGGCAACC TGATGAACAT GCGGCTGGCT GAGTACCTCA AGGGCGGCAC CGGGAACTGG 661 CAGCAGGGCT TCGGCCTGCT GACGGTCGAC GGCAAGCACG TCAAGCCCGA GACCGTGCCG 721 ATCATCGGTG GCCGTTTCAC GGTAGACGGT CACACTTGGG AGGTCTGA The amino acid sequence is as shown in SEQ ID No.2: 1 VTERIVVISD TQIPYDDRRA LRAVIRFIGD YQPTRVIHIG DLMDFPQPSR WNKDTRGEFE 61 GSVFKDAEQC KRRFLDPLRT VYVGPVGVHE GNHDERPRTY LAKYAPALAE SRAFHFENLL 121 DFDGYGIEVL PEFYKVAPGW VTTHGHRGQI SLSRIAGNTA LNAARKFGTS VVMGHTHRLG 181 IGSHTEGYGG ISKRVLTGME VGNLMNMRLA EYLKGGTGNW QQGFGLLTVD GKHVKPETVP 241 IIGGRFTVDG HTWEV As Figure 1 shown, the metallophosphatase of mycobacteriophage is encoded by gene gp48 .

[0028] As Figure 2 shown, the PCR verification result of the inducible expression strain. When performing PCR verification, the amplified product band is between 750 bp and 1000 bp, which is consistent with the size of the target gene, 768 bp.

[0029] Example 2: A method for preparing a bactericidal protein of mycobacteriophage, comprising the following steps: S1: Construct an inducible expression strain of phage gene gp48 ; S2: Measure the growth of the phage gene expression strain; S3: Construct an inducible expression strain of phage metallophosphatase; S4: Purify the phage metallophosphatase protein.

[0030] The specific steps for constructing the inducible expression strain of phage gene gp48 include: S11: Design a primer pair for gene gp48 using the genome of phage A10ZJ24 as a template and perform amplification; S12: Obtain the recombinant plasmid pJR962 and transform it into mycobacteria; S13: Perform PCR verification.

[0031] The primer pair for gene gp48 is: gp48-F: ATAGAGAAGGCGGTATCGGTGACCGAGCGCATCGTCGT; gp48-R: CTTAGCTAATCAGCGGCCGCTCAGACCTCCCAAGTGTGACCGTCT.

[0032] The specific method for obtaining the recombinant plasmid pJR962 is as follows: Using seamless cloning technology, react at 50 °C for 30 minutes to ligate the target fragment to the ClaI-NotI site on the linearized pJR962 induction expression vector, thereby obtaining the recombinant plasmid pJR962.

[0033] Construction and verification of the phage gene induction expression strain: Using the A10ZJ24 phage genome as a template, design primer pairs and amplify them by polymerase chain reaction (PCR). Using seamless cloning technology (Gibson Assembly), react at 50 °C for 30 minutes to ligate the target fragment to the linearized pJR962 induction expression vector (ClaI-NotI site) to obtain a recombinant plasmid. The recombinant plasmid is electrotransformed into wild-type Ms and Mtb strains, and then spread on a 7H10 medium plate containing kanamycin (Kan), and cultured at 37 °C for 3 days (Ms) or 21 days (Mtb). Pick colonies and transfer them to 7H9 medium containing Kan and OADC, and culture at 37 °C. After the logarithmic phase, perform PCR verification.

[0034] Determination of the effect of phage gene expression on bacterial growth: Re-inoculate the verified induction expression strain into 7H9 medium containing Kan, add 100 ng / mL of anhydrous tetracycline (ATc) for induction, and culture in a shaker at 37 °C. Sampling is performed every 4 hours. If it is the Mtb strain, sampling is performed every 1 day. Measure OD600 to observe whether there are growth differences compared with the control strain.

[0035] As Figure 3 shown, Mycobacterium tuberculosis gp48 The colonies of the induction expression strain on the 7H10 solid plate containing 20 ng / mL concentration of ATc are significantly smaller than those of the control strain.

[0036] As Figure 4 shown, Mycobacterium tuberculosis and Mycobacterium smegmatis gp48 The growth inhibition of the induction expression strain in 7H9 medium is significantly enhanced with the increase of ATc concentration.

[0037] Example 3: The specific steps for constructing the phage metallophosphatase induction expression strain include: S31: Using the A10ZJ24 phage genome as a template, design primer pairs for the expression of phage metallophosphatase and amplify the gene gp48; S32: Obtain the recombinant plasmid pET28a-SUMO and transform it into Escherichia coli; S33: Perform PCR verification and sequencing verification.

[0038] The primer pairs for the expression of phage metallophosphatase are as follows: Gp48Exp-F: GGATCCGTGACCGAGCGCATCGTCGT; Gp48Exp-R: AAGCTTTCAGACCTCCCAAGTGTGAC.

[0039] Construction and verification of the strain for the induced expression of phage metallophosphatase: Using the genome of phage A10ZJ24 as a template, after designing the primer pairs, the gene was amplified by PCR gp48 , and after digestion and purification, the fragment was ligated to the BamHI / HindIII sites of the pET28a-SUMO expression vector using T4 ligase to obtain the recombinant plasmid; the recombinant plasmid was transformed into E.coli ArcticExpress (DE3)pRARE2 (WEIDI) expression strain, and spread on an LB solid plate containing kanamycin (Kan), chloramphenicol (Cl), and gentamicin, and cultured overnight at 37 °C; after the colonies formed, they were picked into an LB liquid medium containing triple antibiotics and cultured to the logarithmic phase, and the bacterial liquid was taken for PCR and sequencing verification.

[0040] As Figure 5 shown, the PCR verification result of the strain for the induced expression of metallophosphatase. When performing PCR verification, the amplified product band was between 750 bp and 1000 bp, which was consistent with the size of the target gene, 768 bp.

[0041] Example 4: Purification of the phage metallophosphatase protein includes the following steps: S41: Inoculate the verified correct strain into the medium for culture, centrifuge, and collect the bacterial cells; S42: Break, load onto the column, elute the collected bacterial cells, and obtain the target protein; S43: Detect the target protein, perform dialysis, and then store it.

[0042] When detecting the target protein, the band size and purity are detected by polyacrylamide gel electrophoresis.

[0043] Purification of Bacteriophage Metallophosphatase Protein: Inoculate the verified correct expression strain into 5 mL of LB liquid medium containing triple antibiotics and culture it until the logarithmic growth phase; transfer it to 1 L of LB liquid medium containing triple antibiotics at an inoculation amount of 1% and culture it until OD600 = 0.6 - 0.8. Add 0.3 mM / L of isopropyl-β-D-thiogalactoside (IPTG), induce it at 16°C and 160 rpm for 16 h, and centrifuge at 9000 rpm for 5 minutes to collect the bacteria. Resuspend the collected bacteria with cell lysate (50 mM / L Tris-Base, 50 mM / L NaCl, 5 mM / L imidazole, 10% glycerol, PH = 8.5), sonicate it at 200 W for 30 minutes, and centrifuge at 8000 rpm for 30 minutes to remove cell debris. Incubate the supernatant of the cell lysate with magnetic beads 2+ with Ni at 4°C for 2 hours, and transfer it to a gravity column for elution. Gradient elute the column with elution buffers (50 mM / L Tris-Base, 50 mM / L NaCl, 1 M / L imidazole, 10% glycerol, PH = 8.5) diluted 20-fold (20×), 10-fold (10×), and 2-fold (2×) until the target protein is obtained. Detect the band size and purity of the collected protein by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transfer the protein to a dialysis bag with a pore size of 3.5 kDa and place it in 1 L of dialysis solution (20 mM / L Tris-Base, 50 mM / L NaCl, 20% glycerol, PH = 8.5) for overnight dialysis at 4°C. Filter the dialyzed protein through a 0.2 μm bacterial filter, freeze it in liquid nitrogen and transfer it to a -80°C refrigerator for storage.

[0044] As Figure 6 shown, the polyacrylamide gel electrophoresis result diagram of the purified metallophosphatase protein. The protein band after purification is between 40 kDa and 53 kDa, which is consistent with the size of the target protein of 42 kDa, and there are no obvious impurity bands.

[0045] As Figure 7 shown, the phosphodiesterase activity assay result diagram of the metallophosphatase protein. Each milligram of the protein can produce 210 micromoles of p-nitrophenol (pNP) per minute.

[0046] The above are only the preferred embodiments of the present invention, and do not impose any formal limitations on the present invention. Any simple modifications and equivalent changes made to the above embodiments based on the technical essence of the present invention all fall within the protection scope of the present invention.

Claims

1. A mycobacteriophage bactericidal protein, characterized in that, Including genes gp48 , the gene gp48 has a base sequence as shown in SEQ ID No.

1.

2. The mycobacteriophage bactericidal protein according to claim 1, wherein, The amino acid sequence is shown in SEQ ID No.

2.

3. A method for preparing a mycobacteriophage bactericidal protein, characterized in that, It includes the following steps: S1: Construct a phage gene gp48 induced expression strain; S2: Determine the growth of the phage gene expression strain; S3: Construct a phage metallophosphatase inducible expression strain; S4: Purify the phage metallophosphatase protein.

4. The preparation method of the mycobacteriophage bactericidal protein according to claim 3, characterized in that, The constructed phage gene gp48 The specific steps for inducing the expression strain include: S11: Design gene primers using the A10ZJ24 phage genome as a template and perform amplification; gp48 ​ S12: Obtain the recombinant plasmid pJR962 and transform it into mycobacteria; S13: Conduct PCR verification.

5. The preparation method of the mycobacteriophage bactericidal protein according to claim 4, characterized in that, The gene gp48 has the following primer pair: gp48-F: ATAGAGAAGGCGGTATCGGTGACCGAGCGCATCGTCGT; gp48-R: CTTAGCTAATCAGCGGCCGCTCAGACCTCCCAAGTGTGACCGTCT.

6. The preparation method of the mycobacteriophage bactericidal protein according to claim 4, characterized in that, The specific method for obtaining the recombinant plasmid pJR962 is as follows: Use seamless cloning technology to react at 50 °C for 30 minutes, and ligate the target fragment to the ClaI-NotI site on the linearized pJR962 inducible expression vector to obtain the recombinant plasmid pJR962.

7. The preparation method of the mycobacteriophage bactericidal protein according to claim 3, wherein, The specific steps for constructing the phage metallophosphatase inducible expression strain include: S31: Using the A10ZJ24 phage genome as a template, design a primer pair for phage metallophosphatase expression and amplify the gene gp48 ; S32: Obtain the recombinant plasmid pET28a-SUMO and transform it into Escherichia coli; S33: Conduct PCR verification and sequencing verification.

8. The preparation method of the mycobacteriophage bactericidal protein according to claim 7, characterized in that, The primer pair for the phage metallophosphatase expression is: Gp48Exp-F: GGATCCGTGACCGAGCGCATCGTCGT; Gp48Exp-R: AAGCTTTCAGACCTCCCAAGTGTGAC.

9. The preparation method of the mycobacteriophage bactericidal protein according to claim 3, characterized in that, The purification of the phage metallophosphatase protein includes the following steps: S41: Inoculate the verified correct strain into a medium for culture, centrifuge, and collect the bacterial cells; S42: Break the collected bacterial cells, load them onto a column, elute, and obtain the target protein; S43: Detect the target protein, perform dialysis, and then store it.

10. The preparation method of the mycobacteriophage bactericidal protein according to claim 9, characterized in that, When detecting the target protein, the band size and purity are detected by polyacrylamide gel electrophoresis.

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